Vincent-Sealy Lois V, Thomas Joanna D, Commander Paul, Salmond George P C
Microbiology (Reading). 1999 Aug;145 ( Pt 8):1945-1958. doi: 10.1099/13500872-145-8-1945.
The dsbA genes, which encode major periplasmic disulfide-bond-forming proteins, were isolated from Erwinia carotovora subsp. carotovora (Ecc) and Erwinia carotovora subsp. atroseptica (Eca), and the dsbC gene, encoding another periplasmic disulfide oxidoreductase was isolated from Ecc. All three genes were sequenced and mutants deficient in these genes were created by marker exchange mutagenesis. The Ecc mutants were severely affected in activity and secretion of pectate lyase, probably due to the absence of functional PelC, which is predicted to require disulfide bond formation to achieve its correct conformation prior to secretion across the outer membrane. Similarly, endopolygalacturonase, also predicted to possess disulfide bonds, displayed reduced activity. The major Ecc cellulase (CelV) does not contain cysteine residues and was still secreted in dsbA-deficient strains. This observation demonstrated unequivocally that the localization and activity of the individual components of the Out apparatus are independent of disulfide bond formation. Surprisingly, cellulase activity was shown to be increased approximately two- to threefold in the DsbA mutant. This phenomenon resulted from transcriptional up-regulation of celV gene expression. In contrast, transcription of both pelC and peh were down-regulated in dsbA-deficient strains when compared to the wild-type. Protease (Prt) activity and secretion were unaffected in the Ecc dsbA mutant. Prt activity was considerably reduced in the double dsbA dsbC mutant. However Prt was secreted normally in this strain. The Eca dsbA mutant was found to be non-motile, suggesting that disulfide bond formation is essential for motility in this strain. All of the dsb mutants showed reduced tissue maceration in planta. These results suggest that a feedback regulation system operates in Ecc. In this system, defects in periplasmic disulfide bond formation act as a signal which is relayed to the transcription machinery regulating gene expression in diverse ways.
编码主要周质二硫键形成蛋白的dsbA基因，是从胡萝卜软腐欧文氏菌胡萝卜软腐亚种（Ecc）和胡萝卜软腐欧文氏菌黑腐亚种（Eca）中分离得到的，而编码另一种周质二硫键氧化还原酶的dsbC基因则是从Ecc中分离得到的。对这三个基因进行了测序，并通过标记交换诱变创建了这些基因缺失的突变体。Ecc突变体在果胶酸裂解酶的活性和分泌方面受到严重影响，这可能是由于缺乏功能性的PelC，预计PelC在跨外膜分泌之前需要形成二硫键以实现其正确构象。同样，预计也具有二硫键的内切多聚半乳糖醛酸酶活性降低。Ecc主要纤维素酶（CelV）不含半胱氨酸残基，在dsbA缺陷型菌株中仍能分泌。这一观察结果明确表明，外排装置各组分的定位和活性与二硫键的形成无关。令人惊讶的是，在DsbA突变体中纤维素酶活性显示增加了约两到三倍。这种现象是由celV基因表达的转录上调导致的。相比之下，与野生型相比，dsbA缺陷型菌株中pelC和peh的转录均下调。蛋白酶（Prt）的活性和分泌在Ecc dsbA突变体中未受影响。在dsbA dsbC双突变体中Prt活性显著降低。然而，Prt在该菌株中仍能正常分泌。发现Eca dsbA突变体不能运动，这表明二硫键的形成对该菌株的运动至关重要。所有dsb突变体在植物体内的组织浸解能力均降低。这些结果表明，Ecc中存在一种反馈调节系统。在这个系统中，周质二硫键形成的缺陷作为一种信号，以多种方式传递给调节基因表达的转录机制。
